Methods and systems for measuring growth rate in plant or aquatic animal species

ABSTRACT

Method and systems for measuring growth rate in plant or aquatic animal species such as embryonic or adult fish. The methods and systems utilize the measurement of NADH 2  production by detecting a colorimetric and fluorescent shift when a redox indicator such as resazurin is added to a sample. The colorimetric/fluorescent shift is indicative of the reduction of the redox indicator by NADH 2 . The methods and systems of the present invention may be used to predict growth potential of a plant or animal, and measuring the growth rate of said plant or animal may be helpful for identifying and selecting individuals within a group that have greater growth potential. The methods and systems of the present invention may help eliminate the need for special equipment (e.g., for measuring oxygen consumption), decrease variability of measures, and minimize the effects of external factors (feeding/hormonal status).

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/208,433 filed Aug. 21, 2015, the specification(s) of which is/areincorporated herein in their entirety by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.2010-38500-21758 awarded by USDA/NIFA. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to methods for measuring metabolic rate incells, more particularly to methods for measuring NADH₂ concentrationsand production in cells, more particularly to measuring NAHD₂concentrations and production in cells of plants and/or aquatic animalsfor predicting growth potential of said plants and animals.

BACKGROUND OF THE INVENTION

In fish species, growth is positively correlated with energyexpenditure. Current methods of measuring metabolic rate in fish speciesinvolve measuring oxygen consumption, e.g., measuring oxygen consumptionper unit of body weight. However, measuring oxygen consumption requiresspecialized equipment, training so as to limit fish stress, and anunderstanding of factors that influence metabolic rate (e.g., feedingstatus, hormonal status).

The present invention features methods and systems (e.g., acolorimetric/fluorescent methods and systems) for measuring metabolicrate in plants or aquatic animal species (e.g., embryonic fish,zebrafish, tilapia, trout, etc.). For example, the present inventionfeatures measuring over a period of time (e.g., 1 to 72 hours) theproduction of NADH₂, which is a product of metabolism and thus a directindicator of the flux of metabolites through metabolite oxidation. Themethods and systems of the present invention may monitor NADH₂ using aredox indicator (e.g., resazurin/resorufin or other appropriateindicator (e.g., NADH₂ indicator) such as but not limited to tetrazoliumdyes, e.g., MTT, XTT, MTS, WST). The redox indicator is reduced byNADH₂, resulting in a colorimetric and fluorescent shift in solution. Insome embodiments, the redox indicator remains reduced, and therefore canbe used to provide a cumulative measurement of energy expenditure over atime period.

A review of maternal effects in fish populations (Green, 2008, Advancesin Marine Biology 54:1-105) discusses that female identity explained alarge proportion of variation in egg diameter and in hatching length(e.g., the size of the mother could largely predict the size of thehatchling). Heath and colleagues (Heath et al., 1999, Evolution53(5):1605-1611) studied the maternal effect on progeny growth and foundthat by 180 days, there was no detectable difference between the progenythat were initially bigger due to maternal effect and the progeny thatwere not. Thus, using size and weights of females, embryos, orhatchlings, one cannot necessarily predict how large fish will be beyondthe maternal affect time period (e.g., 2 months). It was surprisinglydiscovered that measuring metabolic rate allowed for the prediction ofgrowth potential beyond the time frame associated with the maternaleffect (e.g., to harvest size), and that high metabolic rates wereindicative of high growth potential. Further, in warm-blooded animals,high metabolic rates are associated with slower growth. It wassurprisingly discovered that in fish, high metabolic rates areassociated with high growth. In oysters, inbred, slow growing lines havea higher metabolic rate than outcrossed fast growing families.

The methods and systems of the present invention may be used to assessthe genetic potential for growth of the plant or animal. For example,the methods and systems may be used to predict the growth potential of aplant or animal. Measuring the metabolic rate of said plant or animalmay be helpful for identifying and selecting individuals within a groupthat have greater predicted growth potential, e.g., individuals that aremost likely to grow faster and/or larger. For example, fish with a highmetabolic rate as embryos may weigh more than 30% more at eight monthsas compared to fish that have low metabolic rates as embryos. Themethods and systems of the present invention may also be used tosegregate fast and slow growing fish. These applications may bebeneficial for the aquaculture industry, e.g., hatcheries, fish farms orthe like. For example, without wishing to limit the present invention toany theory or mechanism, it is believed that the methods and systems ofthe present invention, which may allow for selection of geneticallysuperior brood stock, may have a positive impact on profitability giventhat selecting for genetic potential for growth currently has beenlimited by (a) interactions between aggression and growth, (b) inabilityto select in wild-caught brood stock, and (c) the long generationinterval in slow maturing species.

Without wishing to limit the present invention to any theory ormechanism, it is believed that the methods and systems of the presentinvention may help eliminate the need for special equipment (e.g., formeasuring oxygen consumption), decrease variability of measures, andminimize the effects of external factors (feeding/hormonal status).

The present invention is not limited to use in aquatic animal species(e.g., embryonic fish, zebrafish, tilapia, trout, etc., with the abilityto work with tissue explants and/or primary cells).

The present invention may also be used in plants. For example, thepresent invention may be used to test soil, water, and/or fertilizers.In some embodiments, the plants with the best genetics for growth may beselected. In some embodiments, water quality or soil quality isassessed. In some embodiments, the ability of different fertilizers toenhance growth is assessed.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

SUMMARY OF THE INVENTION

The present invention features a method of brood stock separation from apool of individuals (fish, other aquatic organisms, cells or plants). Insome embodiments, the method comprises measuring whole body metabolicrate in embryonic or juvenile organisms, wherein metabolic rate ismeasured using a colorimetric/fluorescent redox assay comprisingintroducing a redox indicator to the cell sample at time T₀, measuring afluorescence value at time T₀, and measuring a fluorescence value attime T₁ after time T₀; and determining the change in fluorescencebetween T₁ and T₀ to determine a metabolic rate. In some embodiments themethod comprises measuring metabolic rate a tissue sample, whereinmetabolic rate is measured using a colorimetric/fluorescent redox assaycomprising introducing a redox indicator to the cell sample at time T₀,measuring a fluorescence value at time T₀, and measuring a fluorescencevalue at time T₁ after time T₀; and determining the change influorescence between T₁ and T₀ to determine a metabolic rate. The methodcomprises separating the fish or plants based on metabolic rate.

The present invention also features a method of brood stock separationfrom a pool of individuals. In some embodiments, the method comprisesmeasuring whole body metabolic rate in a cell sample from eachindividual, wherein metabolic rate is measured using acolorimetric/fluorescent redox assay comprising introducing a redoxindicator to the cell sample at time T₀; measuring a fluorescence valueat time T₀; measuring a fluorescence value at time T₁ after time T₀; anddetermining the change in fluorescence between T₁ and T₀ to determine ametabolic rate; and separating the individuals in the group that had ametabolic rate in the 10% highest metabolic rates of the group from theremaining individuals in the group. In some embodiments, the pool ofindividuals comprises embryonic or juvenile aquatic organisms, cells, orplants. In some embodiments, the aquatic organisms are fish embryos. Insome embodiments, the aquatic organisms are adult fish. In someembodiments, the individuals in the group that had a metabolic rate inthe 5% highest metabolic rates of the group are separated from theremaining individuals in the group.

The present invention also features a method of stock separation of apool of fish or a group of plants, wherein a tissue sample is obtainedfrom each fish of the pool or each plant in the group. In someembodiments, the method comprises measuring metabolic rate in eachtissue sample, wherein metabolic rate is measured using acolorimetric/fluorescent redox assay comprising introducing a redoxindicator to the cell sample at time T₀, measuring a fluorescence valueat time T₀, and measuring a fluorescence value at time T₁ after time T₀;and determining the change in fluorescence between T₁ and T₀ todetermine a metabolic rate; and separating the fish or plants that had ametabolic rate that is in the 10% highest metabolic rates of the pool offish or group of plants from the remaining fish or plants.

In some embodiments, the fish are fish embryos. In some embodiments, thefish are adult fish. In some embodiments, the sample comprises a tissueexplant. In some embodiments, the fish that are in the 5% highestmetabolic rates are separated from the remaining fish. In someembodiments, the redox indicator comprises AlamarBlue®, resazurin, atetrazolium dye, or PrestoBlue®. In some embodiments, T₁ is equal to T₀plus 8 hours. In some embodiments, T₁ is equal to T₀ plus 16 hours. Insome embodiments, T₁ is equal to T₀ plus 24 hours. In some embodiments,measuring a fluorescence value comprises using a fluorescent platereader. In some embodiments, measuring a fluorescence value comprisesobtaining a digital photograph of the cell sample and quantitating red,green, and blue color intensity, wherein the intensities are correlatedwith fluorescence to determine a quantitative measurement of metabolicrate.

The present invention also features a method for predicting growth ofindividual organisms within a group. In some embodiments, the methodcomprises measuring metabolic rate in a cell sample of the organism byperforming a colorimetric/fluorescent redox assay comprising introducinga redox indicator to the cell sample at time T₀, measuring afluorescence value at time T₀, and measuring a fluorescence value attime T₁; determining a metabolic rate by calculating the change influorescence between T₁ and T₀; and comparing the metabolic rates ofeach of the organisms within the group; wherein a first organism with ahigher metabolic rate than a second organism will be bigger, heavier, orboth bigger and heavier at a time beyond a time associated with materialeffect.

In some embodiments, the organism is a plant or aquatic animal. In someembodiments, the aquatic animal is a fish. In some embodiments, theorganism is an embryonic aquatic organism. In some embodiments, theorganism is an adult aquatic organism. In some embodiments, the redoxindicator comprises AlamarBlue®, resazurin, a tetrazolium dye, orPrestoBlue®. In some embodiments, T₁ is equal to T₀ plus any givenduration of time. In some embodiments, T₁ is equal to T₀ plus 16 hours.In some embodiments, T₁ is equal to T₀ plus 24 hours. In someembodiments, measuring a fluorescence or absorbance value is assessedusing a spectrophotometer with or without fluorescent capabilities. Insome embodiments, measuring a fluorescence value comprises obtaining adigital photograph of the cell sample and quantitating red, green, andblue color intensity, wherein the intensities are correlated withfluorescence to determine a quantitative measurement of metabolic rate.In some embodiments, the time beyond the time associated with maternaleffect is harvest time. In some embodiments, the time beyond the timeassociated with maternal effect is 3 months. In some embodiments, thetime beyond the time associated with maternal effect is 8 months.

The present invention also features a method for assessing geneticpotential for growth of individual organisms within a group. In someembodiments, the method comprises measuring metabolic rate in a cellsample of each of the organism by performing a colorimetric/fluorescentredox assay comprising introducing a redox indicator to the cell sampleat time T₀, measuring a fluorescence value at time T₀, and measuring afluorescence value at time T₁; determining a metabolic rate bycalculating the change in fluorescence between T₁ and T₀; and comparingthe metabolic rates of each of the organisms within the group; wherein afirst organism with a higher metabolic rate than a second organism has ahigher potential for growth than does the second organism.

In some embodiments, the redox indicator comprises AlamarBlue®,resazurin, a tetrazolium dye, or PrestoBlue®. In some embodiments, T₁ isequal to T₀ plus 8 hours. In some embodiments, T₁ is equal to T₀ plus 16hours. In some embodiments, T₁ is equal to T₀ plus 24 hours. In someembodiments, measuring a fluorescence value comprises using afluorescent plate reader. In some embodiments, measuring a fluorescencevalue comprises obtaining a digital photograph of the cell sample andquantitating red, green, and blue color intensity, wherein theintensities are correlated with fluorescence to determine a quantitativemeasurement of metabolic rate.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the present invention will becomeapparent from a consideration of the following detailed descriptionpresented in connection with the accompanying drawings in which:

FIG. 1 shows growth rates of embryos segregated into high and lowmetabolic rate quartiles. *P<0.05.

FIG. 2 shows body length vs. metabolic rate. Body length is similar inhigh and low metabolic rate embryonic fish on the day the assay isperformed.

FIG. 3A-3D show that changes in fluorescence is highly correlated withcaudal fin (FIG. 3A) and skeletal muscle (FIG. 3B) explant mass. Changesin fluorescence increase with duration of incubation with caudal fin(FIG. 3C) and skeletal muscle (FIG. 3D) explants.

FIG. 4A-4B relate to isolated cells from skeletal muscle and fin cellexplants. FIG. 4A: Skeletal muscle satellite cells differentiate intoproliferating myoblasts. FIG. 4B: Using caudal fin derived cells, thepercent confluence was varied to create a range of total metabolic rateswithin a well. Fluorescent change from baseline, increased with # of fincells in a well (P<0.0001).

FIG. 5 shows an analysis of RGB spectrum from wells (G3-G10) in anexperiment using AlamarBlue® reduction methods (pink, purple and bluewells were indicative of embryos with high, moderate, and low metabolicrates, respectively.) Care was taken to ensure measurement was takenneither in a shadow nor ray of light. This preliminary analysis suggeststhat digital photograph analysis of red spectrum intensity can be usedto quantitatively sort wells based on color change. Colors of dots foreach well represent the exact RGB spectrum from the analysis.

FIG. 6 shows a histogram displaying the frequency distribution ofmetabolic rates measured by change in fluorescence. Note the long righttail composed of fish that have a high selective differential from themean.

FIG. 7 shows comparisons between high and low metabolic rate fish madeat identical weights. Slope within metabolic rate group will determinethe relationship between current body weight and explant metabolic rate.Body weight data is from fish that are already selected; thus, selectionof fish with similar masses in each group may be feasible.

FIG. 8A-8C show the growth advantage in tilapia that had a highmetabolic rate (top quartile) as embryos relative to those embryos thathave a low metabolic rate (bottom quartile). The percent increase ingrowth attributable to the high metabolic rate is show at each timepoint for each group (FIG. 8A is group 1, FIG. 8B is group 2, and FIG.8C is group 3). At all time points significant differences existed.

FIG. 9A-9C show AlamarBlue reduction results in lettuce seeds. Lettuceseeds were sprouted in either fertilized or distilled water thentransferred to either fertilized or distilled water 3 days later foranalysis of AlamarBlue reduction. Plants placed into fertilized watershowed increased AlmarBlue reduction at both 3 hours (FIG. 9A) and 24hours (FIG. 9B) relative to plants that were placed into distilledwater. Of note those plants that were sprouted in distilled water didgenerate significantly less signal in fertilized water during the first3 hours of incubation than lettuce sprouts that were always infertilized water. Also shown in FIG. 9C is signal increase with durationof exposure of the lettuce sprout to AlamarBlue, which is an indicationof the cumulative nature of this assay. This figure suggests that thisassay can be used to test the nutrient quality of water or soil.

FIG. 10A-10B show representative results from Oyster Spat. FIG. 10Ashows that oyster spat derived from different families induced nearly 3fold differences in AlamarBlue reduction. This suggests that significantvariation in NADH₂ production exists between families. FIG. 10B showsthat if oyster spat within a cross are size separated those spat thatgrow more slowly (runts; R) induce less signal that oysters that growmore quickly.

FIG. 11A-11D show that in both sized (FIG. 11A, FIG. 11B) and runtoysters (FIG. 11C, FIG. 11D) the signal generated increases with timeand with the number of oysters within a well. Larger oysters (sized)generate signal more quickly and with fewer oysters than do runtoysters.

FIG. 12 shows D-larvae (2-day post fertilization larval oysters)increase the signal generated in this AlamarBlue based assay linearly asthe number of larvae within a well increases (R²=0.9752).

FIG. 13A-13D show changes in fluorescence with various numbers oftilapia (FIG. 13A), trout (FIG. 13B), oysters (FIG. 13C), and shrimp(FIG. 13D) in a set volume. FIG. 13A-13D show that metabolic rate withina well (given volume) increases as the number of individuals within thatwell (volume) increases.

FIG. 14 shows that tilapia broodstock selection based on metabolic rate,as measured by the AlamarBlue based assay, results in offspring withhigher feed efficiency than offspring from Tilapia broodstock selectionbased on growth.

FIG. 15 shows the variability in metabolic rate within a family. Thisproposes that broodstock selection based on family selection will notimprove genetics for growth nearly as rapidly as selection based on theindividual metabolic rate.

FIG. 16 shows that slow growing inbred oyster lines (adam×adam) and(eve×eve) have higher metabolic rates than their outcrossed fastergrowing half-siblings (adam×eve).

DETAILED DESCRIPTION OF THE INVENTION

The present invention features methods and for measuring growth rate inplant or aquatic animal species. The methods and systems of the presentinvention may be used to predict growth potential of a plant or animal,and measuring the growth rate of said plant or animal may be helpful foridentifying and selecting individuals within a group that have greatergrowth potential, e.g., individuals that are most likely to grow fasterand/or larger.

The methods for measuring growth rate (and/or for predicting growthpotential) comprise measuring (over a period of time) the production ofNADH₂ using a redox indicator (e.g., resazurin/resorufin or otherappropriate indicator). The redox indicator is reduced by NADH₂,resulting in a colorimetric and fluorescent shift in solution (e.g.,using AlamarBlue® reduction, pink, purple and blue wells may beindicative of embryos with high, moderate, and low metabolic rates,respectively). The redox indicator remains reduced, and therefore can beused to provide a cumulative measurement of energy expenditure over atime period. Redox indicators may include but are not limited toresazurin, a tetrazolium dye (e.g., MTT, XTT, MTS, WST), PrestoBlue®, orany other appropriate NADH₂ production indicator.

FIG. 1 shows that embryonic fish metabolic rate may be used to predictfuture growth. Further, if fish (e.g., tilapia) are segregated bymetabolic rate, fish with a metabolic rate in the highest quartile growfaster than fish with a metabolic rate in the lowest quartile. FIG. 1also suggests that high metabolic rate fish maintain a growth advantagefor at least 8 months. In some embodiments, the present invention can beused to predict growth at 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, etc. In some embodiments, the presentinvention can be used to predict growth of the animal, e.g., fish, atharvest size/time. In some embodiments, the present invention can beused to predict growth of the animal, e.g., fish, at a time after thematernal effect time period has passed.

Regarding FIG. 1, studies on growth were conducted in three cohorts ofhigh and low metabolic rate fish (n=3). Each cohort included measurementof metabolic rate in at least 6,000 embryonic fish so that eachmetabolic rate group included 1500 fish/experimental unit (n). Becausethese fish were fed identically, this increased growth is alsoindicative of improved feed conversion. Importantly, body length ofthese embryonic fish does not differ on the day and time that theAlamarBlue assay was completed (see FIG. 2). Thus, fish that are largerat the time were not selected for when the assay was performed.

An example of a method for measuring metabolic rate (e.g., metabolicrate assay) is as follows: Embryonic tilapia are rinsed 3 times insterile 28° C. fish water. Using a disposable plastic pipette, embryonicfish are individually transferred into wells of a 96-well plate. Onceplated, 96-well plates containing fish are put into an incubator tomaintain a water temperature of 28° C. Upon completion of plating, allwater is removed from the well and is replaced with 300 μl sterilefiltered assay medium (Fish system water including, 4 mM NaHCO3, 0.1%DMSO, and 0.16% AlamarBlue (Cat. # Y00010; Thermo Fisher ScientificInc.; Waltham, Mass.)). Fluorescence is determined using a fluorescentplate reader (excitation wavelength 530 and emission wavelength 590) atthe beginning of the assay to establish a baseline for each embryo andat the end of the assay (e.g., at 16 h). A large change in fluorescenceis indicative of robust NADH₂ production and a high metabolic rate,while a small change in fluorescence is indicative of a low metabolicrate.

The methods and systems of the present invention are not limited to theaforementioned example. Note that it may be possible for a singleinvestigator to perform the metabolic rate assay on approximately 2,500embryonic fish/8 hr day.

Although selection of brood stock as embryos may work well in captivebred fish with short generation intervals, application in either wildcaught brood stock or slow maturing fish species requires the ability toassess the genetic potential for growth of adult fish. Preliminarystudies establish that the methods and systems of the present invention(e.g., redox assay using AlamarBlue) may be used to assess the metabolicrate of skeletal muscle or caudal fin clip explants. FIG. 3A and FIG. 3Bshows that within samples collected from the same fish, the change influorescence is directly related to the size of the fin or skeletalmuscle explant. Moreover, like previously observed in embryonic fish,explants increase the signal generated with time (see FIG. 3C, FIG. 3D).

Epithelial cells from fin explants and satellite cells from skeletalmuscle have been isolated. FIG. 4A shows the satellite cells fromskeletal muscle can differentiate into proliferating myoblasts. FIG. 4Bshows that AlamarBlue can measure changes in fish fin cell densitywithin a well. Without wishing to limit the present invention to anytheory or mechanism, it is believed that in vitro metabolic rate ofskeletal muscle satellite cell derived myoblasts and caudal finepithelial cells expressed as change in fluorescence per μg DNA may helpsegregate fish with high and low metabolic rates.

As previously discussed, the present invention features methods forpredicting growth in an organism (e.g., a plant, aquatic animal, e.g.,fish, embryonic fish, adult fish, etc.). In some embodiments, the methodcomprising measuring the metabolic rate in a cell sample of theorganism. This may be done by performing a colorimetric/fluorescentredox assay comprising introducing a redox indicator (e.g., resazurin)to the cell sample at time T₀, measuring a fluorescence value at timeT₀, and measuring a fluorescence value at time T₁. The change influorescence between T₁ and T₀ can be calculated to determine themetabolic rate. A high metabolic rate is indicative of a high growthrate, and a low metabolic rate is indicative of a low growth rate.

The redox assay may be performed over a period of time such as 2 hours,4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 20hours, 24 hours, etc. As such, in some embodiments, T₁ is equal to T₀plus 8 hours, T₀ plus 16 hours, T₀ plus 24 hours, etc. The presentinvention is not limited to these times.

Fluorescence may be determined in a variety of ways. For example, insome embodiments, fluorescence is calculated using a fluorescent platereader. In some embodiments, absorbance/fluorescence is calculated usinga digital photograph of the cell sample. In some embodiments, the red,green, and blue color intensity is quantitated. The intensities may becorrelated with fluorescence to determine a quantitative measurement ofmetabolic rate.

The present invention also features a method for brood stock selection.A sample (e.g., cell sample) may be obtained from each fish (e.g., fishembryos, adult fish, etc.). In some embodiments, the method comprisesmeasuring the metabolic rate in each tissue sample and comparing themetabolic rates of each fish in the pool. Particular fish may beselected based on their metabolic rate. For example, in someembodiments, fish that have a metabolic rate that is in the top 50%, top40%, top 30%, top 20%, top 10%, etc. may be selected.

The present invention also features selection based on metabolic ratethrough computer aided red-green-blue (RGB) analysis of digitalphotographs (see FIG. 5.)

Because selection for the genetic potential for growth has been limitedby 1) interactions between aggression and growth, 2) inability to selectin wild-caught brood stock, and 3) the long generation interval in slowmaturing species, the methods of the present invention may allow forselection of genetically superior brood stock, which may have a positiveimpact on profitability of the aquaculturalist. FIG. 5 shows thatmetabolic rate has a distribution with a right extending tail; thus, itmay be possible to impose a large selection differential to maximize theresponse to selection based on metabolic rate. In those industries thatare dependent on wild caught brood stock, the methods of the presentinvention may be used first to allow for selection. In late maturingspecies (e.g., sturgeon), by increasing growth it may be possible toshorten the duration to maturation.

FIG. 6 shows a histogram displaying the frequency distribution ofmetabolic rates measured by change in fluorescence. Note the long righttail composed of fish that have a high selective differential from themean. FIG. 7 shows comparisons between high and low metabolic rate fishmade at identical weights. Slope within metabolic rate group willdetermine the relationship between current body weight and explantmetabolic rate. Body weight data is from fish that are already selected;thus, selection of fish with similar masses in each group may befeasible. FIG. 8A-8C show the growth advantage in tilapia that had ahigh metabolic rate (top quartile) as embryos relative to those embryosthat have a low metabolic rate (bottom quartile). The percent increasein growth attributable to the high metabolic rate is show at each timepoint for each group. At all time points significant differencesexisted.

As previously discussed and as shown in FIG. 9A-9C, the presentinvention may also be used in plants. For example, the present inventionmay be used to test soil, water, and/or fertilizers. In someembodiments, the plants with the best genetics for growth may beselected. In some embodiments, water quality or soil quality isassessed. In some embodiments, the ability of different fertilizers toenhance growth is assessed.

Example 1: Monitoring NADH₂ Production by Adult Cells

Example 1 is an example of the use of the methods and systems of thepresent invention. The present invention is not limited to the examplesset forth in Example 1.

Example 1 describes assessing metabolic rate in adult fish, e.g.,assessing the metabolic rate of tissue explants/cells collected fromadult fish that were segregated by metabolic rate as embryos. This maybe used as a means of selecting for captive bred species. For example,the present invention may be used to assess the ability to ascertaingenetic potential for growth of adult fish by measuring metabolic rateusing minimally invasive, non-lethal techniques to collect fin andskeletal muscle samples. Without wishing to limit the present inventionto any theory or mechanism, it is believed that the methods and systemsof the present invention may be used to evaluate wild caught and slowmaturing brood stock. For example, it is believed that adultcells/explants from fish that had a high metabolic rate as embryos willhave a higher ex vivo metabolic rate than cells/explant from tilapiathat had a low metabolic rate as embryos.

Fin Sampling:

Fish will be weighed then anesthetized in a solution of tricaine methanesulfonate (MS222, 100 mg/L). Mucus will be wiped from the caudal fin.Sterile scissors or a tissue punch will be used to remove a piece of finray from between the bones of the caudal fin. Bacterial contaminationwill be limited by rinsing 3 times for 5 minutes in L-15 media withGentamycin (100 μg/ml), and fungizone (2.5 μg/ml).

Explant Metabolic Rate:

Tissue is plated in 300 μl assay media (L-15 media without phenol redsupplemented with 25 mM HEPES, 5 mM NaHCO3, Penicillin-streptomycin (50I.U./ml), 0.1% DMSO and 0.16% AlamarBlue). Fluorescence is measured attime 0 on a fluorescent plate reader set to excite at 530 nM and measureemission at 590 nM. Explants are incubated in a normal air incubator at28° C. Fluorescence will again be measured at 1, 2, 3, 4, 6, 12, and 24h to measure change across time. At 24 h the tissue will be collected,weighed, and homogenized in lysis buffer (0.1 M phosphate bufferedsaline with 0.1% Triton X-100, PBST) for analysis of DNA using theQuant-iT PicoGreen dsDNA assay kit (Life Technologies, Inc.) to correctall samples for the number of cells. Fluorescence data will be correctedby either tissue weight or total DNA and expressed as change influorescence per mg tissue or μg DNA.

Fin cell isolation will be performed as previously described. Uponreaching confluence in a 3.5 cm petri dish, cells will be plated toconfluence in a 96-well plate and the AlamarBlue assay will be performedas described for explants and corrected for total DNA within the well.

Skeletal Muscle Sampling and Explant/Cell Isolation:

Skeletal muscle samples will be collected by needle (14 G) biopsy in theanesthetized fish from which fin samples were collected. 2-5 mg skeletalmuscle explants will be assayed in triplicate to assess metabolic rateby explant. Changes in fluorescence will be corrected for sample DNA.

Skeletal muscle stem cells (satellite cells) will be isolated from theremainder of the biopsy and grown to confluence. Upon reachingconfluence satellite derived proliferating myoblasts will be plated intoa 96-well plate and the AlamarBlue assay will be performed as describedfor fin cells. Alternatively, myoblasts may be stimulated to formnascent muscle fibers so that metabolic rate can be measured on thedifferentiated cell type.

Without wishing to limit the present invention to any theory ormechanism, it is believed that samples collected from fish that weredetermined to have a low metabolic rate as embryos will maintain a lowermetabolic rate than samples collected from fish that had a highmetabolic rate as embryos. FIG. 3 and FIG. 4B show that metabolic ratein explants and isolated cells can be accurately measured. It isbelieved that the metabolic rate from both skeletal muscle and finsamples can be accurately assessed. And, it is believed that themetabolic rate assessed from skeletal muscle samples will correlate withmetabolic rate assessed from fin samples.

Data may be analyzed in SAS. The effect of embryo metabolic rate (highor low), adult body weight, and their interaction on metabolic rate fromexplants or cell will be assessed for each tissue type using a mixedmodel two-way ANOVA in SAS (SAS Inc., Cary, N.C.). Correlation analysiswill be used to assess relationships between metabolic rate assessed bytissue type and sample type (cells or explants; Proc Corr). Regressionanalyses will be performed to quantitate this effect as needed.

The present invention may also be used to assess selection of broodstock based on metabolic rate of tissue samples, e.g., metabolic rate ofexplants from adult fish may be indicative of offspring metabolic rate.Future work may focus on this application in wild-caught brood stock.

Methods:

Brood stock rearing and selection: 300 fish will be grown in a startertank and moved as needed to maximally encourage growth and development.Feed will be provided 3 times daily to satiation. At 4 months of age,skeletal muscle or fin biopsies will be collected for metabolic ratedetermination as described and validated in Experiment 2. Fish will betagged for identification and upon determination of metabolic rate, fishin the top and bottom 10% will be isolated and moved to otherfacilities.

Breeding:

Brood stock in the high and low metabolic rate groups will be dividedinto five tanks with 6 brood stock in each tank (n=5). The mouths offemale fish will be checked every week for embryonic fish. Embryos willbe collected and assayed for metabolic rate as previously described. Toprevent continuously sampling from the same female fish, a female thatprovides a clutch will be immediately removed from the study. Studieswill continue until at least 2 clutches have been collected and analyzedfrom each tank.

A one-way ANOVA will be used to assess the effect of explant/cellmetabolic rate on embryo metabolic rate. Tank will serve as theexperimental unit (n=5). Accordingly, the single measurement of eachembryo will be nested within brood and brood nested within tank. Powercalculation mirrors that from experiment 1.

Without wishing to limit the present invention to any theory ormechanism, it is believed that the metabolic rate of explants from adultfish will be positively related to the offspring metabolic rate. Thisresult would indicate that brood stock can be selected based onmetabolic rate of adult tissue samples.

Example 2: Field Application

Example 2 is an example of the use of the methods and systems of thepresent invention. The present invention is not limited to the examplesset forth in Example 2.

Example 2 describes procedures that allow for field application of themethods and systems of the present invention. For example, Example 2describes assessing results of methods of the present invention using acontrolled digital photograph, eliminating the requirement for afluorescent spectrophotometer.

First, the quantitative assessment of color change from digitalphotographs will be compared to that obtained using a fluorescent platereader, e.g., quantitated RGB color will be correlated with fluorescentsignal measured on a plate reader. Without wishing to limit the presentinvention to any theory or mechanism, it is believed that the use ofdigital photographs may be an adequate substitute for a fluorescentplate reader, allowing the methods of the present invention to beperformed in the field.

Methods:

Plates are digitally captured using a 12 megapixel digital camera. Inall pictures, a standard white background is used and a color referencechart is included to correct for potential differences in lighting.Using Adobe Photoshop eyedropper tool, the red, green, and blue colorwithin each well will be quantitated (see FIG. 5, preliminary data). Thered, green, and blue color intensity, differences between intensity ofdifferent colors (e.g. Red-Green), and ratios of color intensity (e.g.Red/Blue) are correlated with fluorescence to identify the colorspectrum quantitation that best allows for quantitative analysis ofmetabolic rate from a color photo. Data may first be analyzed through XYplots using GraphPad Prism version 5.00 for Windows, GraphPad Software,San Diego Calif. USA, www.graphpad.com to visually understand therelationship between fluorescence and each variable of color (intensityof a single color, differences, ratios). It is possible that linearrelationships will be found, and thus Proc Corr in SAS (SAS Institute,Cary, N.C.) will then be performed to identify the factor with thegreatest correlation with change in fluorescence. Proc Reg in SAS may beused to quantitate the relationship between fluorescence and quantitatedRGB spectrum. If fluorescence and RGB values are not linearly relatedthe data may be transformed (or a non-linear regression analysis may beperformed).

Next, the possibility of shipping explants for analysis in a central labwill be assessed. Without wishing to limit the present invention to anytheory or mechanism, it is believed that explants may retain theirmetabolic rate (e.g., fluorescence per mg tissue) for a period of time,e.g., 12 hr or more, after collection. Maintaining explants at 4° C. mayextend viability.

Methods:

The differences in the timing of assay initiation and sample incubationtemperature will be compared to assess the possibility that explantscould be collected on farm and shipped to a commercial laboratory foranalysis. The present invention establishes that embryonic fish andoysters can be collected and shipped to a central lab for analysis, thusit may be possible for explants as well.

Within a fish, the coefficient of variation in metabolic rate ofskeletal muscle and fin explants is low (5.7 and 6.2%, respectively). Bycollecting multiple samples from the same individuals, it may bepossible to perform comparisons to thoroughly analyze the effects ofassay timing and incubation temperatures on explant viability. Thisstudy will be conducted testing 5 different times (0, 6, 12, 24, and 48h) and 2 different incubation temperatures (4 or 22 degrees C.). Eachcondition will be run in triplicate within a fish. As such, 30samples/fish are needed. To accommodate the need for this large numberof samples/fish, samples will be collected immediately post-mortem fromfish anesthetized in an ice water slurry and sacrificed by decapitation.Each sample will be placed in a capped culture tube containing 1 ml L-15medium without phenol red supplemented with 25 mM HEPES, 5 mM NaHCO3,Gentamycin (100 μg/ml), and fungizone (2.5 μg/ml). Three samples fromeach fish will be exposed to each condition. Samples maintained at bothroom temperature and at 4° C. will be kept in Styrofoam shippingcontainers within the lab and samples will be removed at 6, 12, 24, and48 hours of incubation. At the end of the incubation samples will beanalyzed as previously described for metabolic rate. At the conclusionof the study, explants will be weighed and total DNA within the samplewill be assessed. Fluorescence data will be corrected by either tissueweight or total DNA and expressed as change in fluorescence per mgtissue or μg DNA.

Example 3: Additional Experiments

FIG. 10A-10B show representative results from Oyster Spat. The topfigure shows that oyster spat derived from different families inducednearly 3 fold differences in AlamarBlue reduction. This suggests thatsignificant variation in NADH₂ production exists between and withinfamilies. In the middle figure we can see that if oyster spat within across are size separated those spat that grow more slowly (runts; R)induce less signal that oysters that grow more quickly. FIG. 11A-11Dshow that in both sized and runt oysters the signal generated increaseswith time and with the number of oysters within a well. Larger oysters(sized) generate signal more quickly and with fewer oysters than do runtoysters. FIG. 12 shows D-larvae (2-day post fertilization larvaloysters) increase the signal generated in this AlamarBlue based assaylinearly as the number of larvae within a well increases (R²=0.9752).FIG. 13A-13D show changes in fluorescence with various numbers oftilapia (FIG. 13A), trout (FIG. 13B), oysters (FIG. 13C), and shrimp(FIG. 13D). FIG. 13A-13D show that metabolic rate within a given volumeincreases as the number of individuals within that volume increases.FIG. 14 shows that tilapia broodstock selection based on metabolic rate,as measured by the AlamarBlue based assay, results in offspring withhigher feed efficiency than offspring from Tilapia broodstock selectionbased on growth. FIG. 15 shows the variability in metabolic rate withina family. This proposes that broodstock selection based on familyselection will not improve genetics for growth nearly as rapidly asselection based on the individual metabolic rate. FIG. 16 shows thatslow growing inbred oyster lines (adam×adam) and (eve×eve) have highermetabolic rates than their outcrossed faster growing half-siblings(adam×eve).

REFERENCES

The disclosures of the following documents are incorporated in theirentirety by reference herein: (1) Gjedrem, T., Aquaculture Research,2000. 31(1): p. 25-33. (2) Conceicao, L. E. C., Y. Dersjant-Li, and J.A. J. Verreth, Aquaculture, 1998. 161(1-4): p. 95-106. (3) Gjerde, B.,Aquaculture, 1986. 57(1-4): p. 37-55. (4) Huang, C. M. and I. C. Liao,Aquaculture, 1990. 85(1-4): p. 199-205. (5) Hulata, G., G. W. Wohlfarth,and A. Halevy, Aquaculture, 1986. 57(1-4): p. 177-184. (6) Tave, D. andR. O. Smitherman, Transactions of the American Fisheries Society, 1980.109(4): p. 439-445. (7) Thodesen, J., et al., Aquaculture, 2011. 322: p.51-64. (8) Gadagkar, S. R., Social behaviour and growth rate variationin cultivated tilapia (Oreochromis niloticus). 1997, DalhousieUniversity: Dalhousie University. (9) Koebele, B., Environmental Biologyof Fishes, 1985. 12(3): p. 181-188. (10) Blanckenhom, W. U., EthologyEcology & Evolution, 1992. 4(3): p. 255-271. (11) Seiler, S. M. and E.R. Keeley, Animal Behaviour, 2007. 74(6): p. 1805-1812. (12) Grant, J.W. A., Canadian Journal of Fisheries and Aquatic Sciences, 1990. 47(5):p. 915-920. (13) Huntingford, F. A., et al., Journal of Fish Biology,1990. 36(6): p. 877-881. (14) McCarthy, I. D., C. G. Carter, and D. F.Houlihan, Oncorhynchus mykiss (Walbaum). Journal of Fish Biology, 1992.41(2): p. 257-263. (15) Allee, W. C., et al., Journal of ExperimentalZoology, 1948. 108(1): p. 1-19. (16) Magnuson, J. J., Canadian Journalof Zoology, 1962. 40(2): p. 313-363. (17) Purdom, C. E., Variation inFish, in Sea Fisheries Research, F. R. H. Jones, Editor. 1974, ElekScience: London. p. 347-355. (18) BROWN, M. E., Journal of ExperimentalBiology, 1946. 22(3-4): p. 118-129. (19) Wohlfarth, G. W., Shoot carp.Bamidgeh, 1977. 29(2): p. 35-56. (20) Clarke, A. and N. M. Johnston,Journal of Animal Ecology, 1999. 68(5): p. 893-905. (21) Miyashima, A.,et al., Aquaculture Research, 2012. 43(5): p. 679-687. (22) Cook, J. T.,A. M. Sutterlin, and M. A. McNiven, Aquaculture, 2000. 188(1-2): p.47-63. (23) Livingston, R. J., Journal of the Marine BiologicalAssociation of the United Kingdom, 1968. 48: p. 485-497. (24) Renquist,B. J., et al., Zebrafish, 2013. 10(3): p. 343-52. (25) Williams, S. Y.and B. J. Renquist, Journal of Visualized Experiments, 2015. In Press.(26) Smith, R. W. and D. F. Houlihan, Journal of Comparative PhysiologyB, 1995. 165(2): p. 93-101. (27) Brand, M. D., et al., Evolution ofenergy metabolism. Proton permeability of the inner membrane of livermitochondria is greater in a mammal than in a reptile. Biochem J, 1991.275 (Pt 1): p. 81-6. (28) El-Greisy, Z. A. and A. E. El-Gamal, TheEgyptian Journal of Aquatic Research, 2012. 38(1): p. 59-66. (29) Siraj,S. S., et al. International Symposium on Tilapia in Aquaculture. 1983.Nazareth, Isreal: Tel Aviv University. (30) Palada-de Vera, M. S. and A.E. Eknath. Proceedings of the Fourth International Symposium on Geneticsin Aquaculture. 1993. Wuhan, Hubei Province, China: Elsevier. (31)Mauger, R. E., P. Y. Le Bail, and C. Labbe, Comparative Biochemistry andPhysiology B-Biochemistry & Molecular Biology, 2006. 144(1): p. 29-37.(32) Vanmeter, D. E., Progressive Fish-Culturist, 1995. 57(2): p.166-167.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

Although there has been shown and described the preferred embodiment ofthe present invention, it will be readily apparent to those skilled inthe art that modifications may be made thereto which do not exceed thescope of the appended claims. Therefore, the scope of the invention isonly to be limited by the following claims. Reference numbers recited inthe claims are exemplary and for ease of review by the patent officeonly, and are not limiting in any way. In some embodiments, the figurespresented in this patent application are drawn to scale, including theangles, ratios of dimensions, etc. In some embodiments, the figures arerepresentative only and the claims are not limited by the dimensions ofthe figures. In some embodiments, descriptions of the inventionsdescribed herein using the phrase “comprising” includes embodiments thatcould be described as “consisting of”, and as such the writtendescription requirement for claiming one or more embodiments of thepresent invention using the phrase “consisting of” is met.

1. A method of brood stock separation from a pool of individuals, saidmethod comprising: a. measuring whole body metabolic rate in a cellsample from each individual, wherein metabolic rate is measured using acolorimetric/fluorescent redox assay comprising introducing a redoxindicator to the cell sample at time T₀; measuring a fluorescence valueat time T₀; measuring a fluorescence value at time T₁ after time T₀; anddetermining the change in fluorescence between T₁ and T₀ to determine ametabolic rate; and b. separating the individuals in the group that hada metabolic rate in the 10% highest metabolic rates of the group fromthe remaining individuals in the group.
 2. The method of claim 1,wherein the pool of individuals comprises embryonic or juvenile aquaticorganisms, cells, or plants.
 3. The method of claim 2, wherein theaquatic organisms are fish embryos.
 4. The method of claim 2, whereinthe aquatic organisms are adult fish.
 5. The method of claim 1, whereinindividuals in the group that had a metabolic rate in the 5% highestmetabolic rates of the group are separated from the remainingindividuals in the group.
 6. The method of claim 1, wherein the redoxindicator comprises AlamarBlue®, resazurin, a tetrazolium dye, orPrestoBlue®.
 7. The method of claim 1, wherein T₁ is equal to T₀ plus 8hours, T₀ plus 16 hours, or T₀ plus 24 hours.
 8. (canceled) 9.(canceled)
 10. The method of claim 1, wherein measuring a fluorescencevalue comprises using a fluorescent plate reader.
 11. The method ofclaim 1, wherein measuring a fluorescence value comprises obtaining adigital photograph of the cell sample and quantitating red, green, andblue color intensity, wherein the intensities are correlated withfluorescence to determine a quantitative measurement of metabolic rate.12. A method for predicting growth of individual organisms within agroup, said method comprising: a. measuring metabolic rate in a cellsample of the organism by performing a colorimetric/fluorescent redoxassay comprising introducing a redox indicator to the cell sample attime T₀, measuring a fluorescence value at time T₀, and measuring afluorescence value at time T₁; b. determining a metabolic rate bycalculating the change in fluorescence between T₁ and T₀; and c.comparing the metabolic rates of each of the organisms within the group;wherein a first organism with a higher metabolic rate than a secondorganism will be bigger, heavier, or both bigger and heavier at a timebeyond a time associated with material effect.
 13. The method of claim12, wherein the organism is a plant or aquatic animal.
 14. The method ofclaim 13, wherein the aquatic animal is a fish.
 15. The method of claim13, wherein the organism is an embryonic aquatic organism.
 16. Themethod of claim 13, wherein the organism is an adult aquatic organism.17. The method of claim 12, wherein the redox indicator comprisesAlamarBlue®, resazurin, a tetrazolium dye, or PrestoBlue®.
 18. Themethod of claim 12, wherein T₁ is equal to T₀ plus any given duration oftime.
 19. (canceled)
 20. (canceled)
 21. The method of claim 12, whereinmeasuring a fluorescence or absorbance value is assessed using aspectrophotometer with or without fluorescent capabilities.
 22. Themethod of claim 12, wherein measuring a fluorescence value comprisesobtaining a digital photograph of the cell sample and quantitating red,green, and blue color intensity, wherein the intensities are correlatedwith fluorescence to determine a quantitative measurement of metabolicrate.
 23. The method of claim 12, wherein the time beyond the timeassociated with maternal effect is harvest time.
 24. The method of claim12, wherein the time beyond the time associated with maternal effect is3 months or 8 months. 25.-32. (canceled)